Compositions and methods for treating hepatitis B

ABSTRACT

The present invention provides compositions and methods for treating hepatitis B virus (HBV) infection as well as methods for identifying a compound or a composition that is suitable for treating HBV infection. In addition, the present invention provides a suitable non-mammalian animal model that can be used to screen for a compound or a composition that can inhibit HBV replication or treat HBV infection in a mammal. In particular, the present invention provides compositions and methods for treating hepatitis B infection by inhibiting interaction between HBV x protein and a Bcl-2 family protein or by reducing the expression level of a Bcl-2 family protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication No. 61/706,083, filed Sep. 26, 2012, which is incorporatedherein by reference in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under grant numbersGM059083, GM079097 and GM088241 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for treatinghepatitis B virus (HBV) infection. In particular, the present inventionrelates to compositions and methods for treating hepatitis B infectionby modulating interaction between HBV x protein and a Bcl-2 familyprotein or by reducing the expression level of a Bcl-2 family protein.

BACKGROUND OF THE INVENTION

Hepatitis B Virus (HBV) infects more than 400 million people in theworld and is the leading cause of hepatocellular carcinoma (HCC) andother liver disease. It has been a major health issue in many countries.Although the current HBV vaccine can prevent new HBV infections, thereis no effective treatment for chronic HBV carriers, many of whom willeventually develop various liver disorders and HCC.

Studies have shown that one of the key pathogenic and oncogenic proteinsencoded by HBV is the 17-kDa HBV x protein (HBx). HBx is amulti-functional HBV protein that has shown to be crucial for HBVinfection and pathogenesis and a contributing cause of hepatocytecarcinogenesis. As appropriately implied by its name, HBx is anenigmatic protein that can bind to a vast number of proteins in variousin vitro systems. Unfortunately, the exact host targets and mechanismsof action of HBx are poorly characterized. In fact, to date it has beena major challenge to identify cellular targets of HBx and its mechanismsof action. Because of HBx's role in HBV infection and pathogenesis, itis believed that identification of specific cellular targets of HBx willprovide important therapeutic potential in treating chronic HBVcarriers.

Accordingly, there is a need to identify cellular target(s) or ligand(s)of HBx. In addition, there is a need for treating HBV infection bytargeting cellular target(s) of HBx.

SUMMARY OF THE INVENTION

Some aspects of the invention are based on the discovery by the presentinventors that human anti-apoptotic proteins, Bcl-2 and Bcl-xL, are hosttargets of HBx. In addition, the present inventors have discovered thatinteractions between HBx and Bcl-2, Bcl-xL, or both are required tostimulate cytosolic calcium elevation, HBV viral replication, and celldeath induction. These discoveries by the present inventors of Bcl-2proteins as the host targets of HBx-induced viral replication and hostcell death are useful in identifying and/or producing compositions forthe treatment of chronic HBV patients.

Studies have shown that patients with high HBV viral DNA titers are morelikely to develop HCC. Thus, in some embodiments of the invention,discoveries by the present inventors can be used to significantly reduceHBV replication, and therefore the likelihood of developing HCC. Infact, discoveries disclosed herein by the present inventors can be usedto treat various clinical conditions associated with HBV infection by,for example, significantly reducing the HBV replication. Exemplaryclinical conditions that can be treated by compositions and methods ofthe invention include, but are not limited to, host inflammation induceby HBV-activated necrosis and apoptosis, cirrhosis, HCC, and hepatitis.

In some embodiments, compositions of the invention bind to the Bcl-2homology 3 (BH3)-like motif (i.e., BH3-domain) of HBx, Bcl-2 or Bcl-xL,or another Bcl-2 family member.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a shows peptaibol TK sequence alignment. The red rectangleshighlight the minor differences among the sequences.

FIG. 1b is a bar graph showing peptaibol TK causes more persistent cellcorpses in ced-1 (e1735) embryos. Stages of embryos examined were comma,1.5-fold, 2-fold, 2.5-fold, 3-fold, and 4-fold. They axis representsaverage number of cell corpses scored and error bars represent SEM.

FIG. 1c is a bar graph showing peptaibol TK causes more persistent cellcorpses in the germline of ced-1(e1735) animals. Germ cell corpses inanimals 24 hour and 48 hour post L4 to the adult molt were scored(n=30).

FIG. 1d is a bar graph showing chemical genetic analysis of thepeptaibol TK target in the core apoptosis pathway in C. elegans.Peptaibol TK treatment was performed on the following cell deathmutants: ced-1(e1735), ced-1(e1735); egl-1(n3082), ced-1(e1735);ced-9(n1950gf), ced-1(e1735); ced-4(n1162), ced-1(e1735); ced-3(n2433),and ced-1(e1735) smIs111; ced-4(n1162). smIs111 is an integratedtransgene carrying P_(egl-1)acCED-3. Two-fold stage embryos were scoredin each experiment.

FIG. 1e is a bar graph showing peptaibol TK can synergize with EGL-1 toinduce apoptosis. ced-1(e1735); egl-1(n3082); smIs82 animals weretreated with peptaibol TK and subjected to heat-shock treatment.Two-fold stage embryos were scored for cell corpses. smIs82 is anintegrated transgene carrying P_(hsp)EGL-1. Data are means±SEM. Morethan 20 embryos (b, d, e) or 30 animals (c) were scored in eachexperiment. ** P<0.01; *** P<0.001.

FIG. 1f is a schematic illustration showing interaction of peptaibol TKwith the core apoptosis pathway in C. elegans. Peptaibol TK likelyinduces and enhances apoptosis upstream of or in parallel to EGL-1.

FIG. 2 is a graph showing peptaibol TK enhances HBx-induced cell deathin C. elegans. Two different HBx transgenic animals, smIs98 and bzIs8;smEx(P_(mec-7)HBx), and two control transgenic animals lacking HBxexpression, smIs3 and bzIs8, were treated with 5 μM of Peptaibol TK.smIs98 is an integrated transgene containing P_(mec-3)GFP andP_(mec-7)HBx. smIs3 is an integrated transgene containing P_(mec-3)GFP,which directs GFP expression in ten C. elegans sensory neurons,including the PLM touch receptor neurons. bzIs8 is an integratedtransgene containing P_(mec-4)GFP, which directs GFP expression in sixC. elegans touch receptor neurons, including the PLM neurons. Thepresence of GFP-positive PLM neurons was scored using a Nomarskimicroscope. The data are shown in three categories: both PLM missing(black bars), one PLM missing (gray bars) and no PLM missing (whitebars). The y axis represents percentage of each category. Percentagenumbers under columns indicate the total PLM missing percentage in eachexperiment. More than 40 animals were scored in each experiment.

DETAILED DESCRIPTION OF THE INVENTION

Infection with the hepatitis B virus (HBV) can lead to a variety ofclinical condition including, but not limited to, the development ofhepatitis, liver inflammation, cirrhosis, and hepatocellular carcinoma(HCC). HBV infection is a leading cause of morbidity and mortalityworldwide. HBV X protein (HBx) is an important effector for HBVpathogenesis, but its cellular targets and acting mechanisms to datehave remained elusive. Disclosed herein is a discovery by the presentinventors that HBx interacts with the anti-apoptotic proteins Bcl-2 andBcl-xL through a Bcl-2 homology 3 (BH3)-like motif in mammalianhepatocytes. The present inventors have also observed that mutations inthe BH3-like motif that prevent HBx binding to Bcl-2 and Bcl-xLabrogated cytosolic calcium elevation and cell death induced by HBxexpression in hepatocytes and severely impaired HBV replication. Thegreatly reduced HBV replication caused by mutations in the BH3-likemotif of HBx can be substantially rescued by restoring cytosoliccalcium. These results show that HBx binding to Bcl-2 family members andsubsequent elevation of cytosolic calcium are important for HBV viralreplication.

Moreover, RNA interference knockdown of Bcl-2 or Bcl-xL resulted inreduced calcium elevation induced by HBx and decreased viral replicationin hepatocytes. These results further indicate that HBx targets Bcl-2proteins through its BH3-like motif to promote cytosolic calciumelevation, cell death, and viral replication during HBV pathogenesis.Accordingly, some aspects of the invention provide methods for treatingHBV infection by (i) inhibiting binding of HBx with a Bcl-2 familyprotein, (ii) reducing the expression of a Bcl-2 family protein; or(iii) a combination thereof.

HBV is a hepatocyte-specific DNA virus, which encodes several differentviral proteins, including DNA polymerase, surface antigen, core antigen,and the X protein (HBx). While the functions of the other viral genes inHBV DNA replication and virion assembly are better understood, the rolesand mechanisms of HBx in HBV infection and pathogenesis remainenigmatic. HBx has been implicated in mediating multiple viral andcellular events in HBV-infected cells, including viral replication,transactivation of transcription factors, signal transduction, cellcycle progression, and cell death. Although HBx is found in both thecytoplasm and the nucleus, mitochondria appear to be an important sitefor HBx action, because expression of HBx has been shown to induceaggregation of mitochondria, loss of mitochondrial membrane potential,and cytochrome c release.

HBx is necessary for viral pathogenesis and oncogenesis in HBV-infectedlivers. The HBx gene has been shown to be one of the most frequentlyintegrated viral sequences in HCC. In fact, HBx protein is detected inmost patients with HBV-related HCC, even in the absence of viral DNAreplication. In some cases, HBV variants carrying mutations in HBx havebeen identified in HCC tissues. Such mutations have resulted in the lossof HBx-dependent activities, indicating that evolving HBx functions mayunderlie HBV-related liver disease. It has also been shown that HBxpromotes liver tumorigenesis in transgenic mice lacking the othercomponents of the HBV virion. Thus, it is clear that HBx plays animportant role in the development of HBV-related HCC.

Calcium signaling has been shown to be important for a variety of HBxactivities. For example, HBx-induced elevation of cytosolic calcium hasbeen shown to be important for HBV DNA replication, HBV core assembly,and activation of several transcriptional events and signaling cascades.Induction of apoptosis or necrosis by HBx also requires increasedcytosolic calcium and mitochondria permeability transition (MPT), aprocess by which mitochondria regulate cellular calcium duringhomeostasis and cell death. However, to date the cellular targets withwhich HBx interacts to induce MPT and cytosolic calcium increase havenot been identified.

Many proteins were found to interact with HBx in various in vitrosystems. However, most of these protein interactions have not beenconfirmed in conditions that recapitulate HBV infection in hepatocytes.Genetic redundancy of complex mammalian systems has been a major hurdleto definitive identification of HBx cellular targets. Using agenetically tractable C. elegans animal model, the present inventorshave discovered that HBx interacts directly with the Bcl-2 homolog,CED-9, to induce cytosolic calcium increase and cell death, mimickingtwo important events downstream of HBx expression in hepatocytes. Inaddition, the present inventors have discovered that HBx interacts withtwo Bcl-2 family members (Bcl-2 and Bcl-xL) in hepatocytes to inducecytosolic calcium elevation, cell death, and viral DNA replication.

The HBV X gene, one of the four coding genes in the HBV genome, encodesa multi-functional protein (HBx) that is essential for HBV infection andreplication. HBx also affects multiple cellular events in infectedcells, including transcription, signal transduction, proteasomeactivity, cell cycle progression, and cell death. HBx has also beenshown to play an important role in neoplastic transformation ofhepatocytes in HBV-infected patients. Even in transgenic mice expressingonly HBx, high incidences of liver tumors were found indicating a causalrelationship between HBx and HCC.

Studies have linked HBx expression to the activation of necrosis andapoptosis in hepatocytes. HBx can sensitize liver cells to cell death byvarious insults, including tumor necrosis factor-α (TNF-α) and growthfactor deprivation. HBx has also been shown to induce mitochondriaaggregation, loss of mitochondrial membrane potential, and cytochrome crelease, indicating that HBx may act through mitochondria to induce celldeath. Apoptosis and necrosis are also early events of liverpathogenesis in HBx transgenic mice and can lead to development ofcirrhosis and HCC. While these results suggest a hepatotoxic functionfor HBx, to date the cellular targets and signaling pathways that HBxexploits to promote cell death and the development of HCC have remainedunclear. Without being bound by any theory, it is believed that chronichepatocyte cell death causes cycles of inflammatory cytokine release,local liver damage, and compensatory regeneration, leading to thecontinual acquisition of oncogenic mutations and the development of HCC.Therefore, identification of cellular targets and pathways that mediateHBx-induced cell death can lead to therapeutically effective treatmentof HBV-related clinical conditions including a variety of liverdiseases.

Calcium signaling has been shown to play an important role in a widevariety of HBx activities. For example, HBx has shown to be responsiblefor triggering cytosolic Ca²⁺ increase in HBV-infected hepatocytes,which is required for HBV DNA replication. HBx-induced elevation ofcytosolic Ca²⁺ is also important for HBV core assembly, someHBx-activated transcriptional events, and activation of severalsignaling cascades, including JNK and MAPK pathways. HBx has also beenshown to modulate cell death by altering cytosolic Ca²⁺. Although it hasbeen suggested that HBx targets mitochondria and mitochondriapermeability transition (MPT) in Ca²⁺ regulation, the cellular target(s)with which HBx interacts to alter cytosolic Ca²⁺ has been unknown untilthe discovery by the present inventors, which is disclosed herein.

Given the complexity of HBx study in mammals, the present inventors haveused a simple, genetically tractable animal model C. elegans to identifyHBx targets and signaling pathways. As disclosed herein, the presentinventors discovered that the human Bcl-2 homolog, CED-9, is thecellular target that HBx interacts with to induce cytosolic Ca²⁺increase and cell apoptosis.

One particular aspect of the invention provides a method for treatinghepatitis B infection in a subject. Such a method comprisesadministering to a subject in need of such a treatment a composition (i)that is capable of inhibiting binding of hepatitis B virus X protein toa Bcl-2 family member protein in the subject; (ii) that is capable ofreducing the expression of Bcl-2 family member protein in the subject;or (iii) a combination thereof. It should be noted that the term“treating” or “treatment” of a disease includes: (1) preventing thedisease, i.e., causing the clinical symptoms of the disease not todevelop in a subject that may be exposed to or predisposed to thedisease but does not yet experience or display symptoms of the disease;(2) inhibiting the disease, i.e., arresting or reducing the developmentof the disease or its clinical symptoms; or (3) relieving the disease,i.e., causing regression of the disease or its clinical symptoms. Theterm “inhibiting” a protein refers to a decrease in the level ormagnitude of an activity or expression of the protein. For example,inhibition of Bcl-2 protein includes reduction in Bcl-2 genetranscription, Bcl-2 gene translation, and/or Bcl-2 protein activity.Inhibition of a particular protein by a composition can be readilydetermined using any of the methods known to one skilled in the art,e.g., by determining any parameter that is indirectly or directlyaffected by the expression of the gene encoding that protein or bydetermining the activity of the protein. Such parameters include, butare not limited to, changes in RNA or protein levels, changes in proteinactivity, changes in product levels, changes in downstream geneexpression, changes in reporter gene transcription (luciferase, CAT,β-galactosidase, β-glucuronidase, green fluorescent protein (see, e.g.,Mistili & Spector, Nature Biotechnology 15:961-964 (1997)); changes insignal transduction, phosphorylation and dephosphorylation,receptor-ligand interactions, second messenger concentrations (e.g.,cGMP, cAMP, IP3, and Ca2+), and cell growth.

In some embodiments, the Blc-2 family member protein comprises Bcl-2,Blc-xL, another Bcl-2 member, or a combination thereof.

Still in other embodiments, the composition comprises a bindinginhibitor that is capable of inhibiting binding of hepatitis B virus Xprotein to said Bcl-2 family member protein. Such binding inhibitor canbe a small organic molecule or a drug, small peptides, small RNAmolecules such as aptamers, antibodies, siRNA (short inhibitory RNA), ora short hairpin RNA (shRNA) that reduces the expression of thecorresponding gene, such as those disclosed in the Examples section. Inaddition, other suitable shRNAs that can inhibit expression of Bcl-2family member protein can be readily determined using the proceduresdescribed herein. Synthesis of such suitable shRNAs is well known and isoften achieved using any of the commercially available automaticoligonucleotide synthesizers. Small organic molecules (e.g., less than 1kD in molecular weight) that can inhibit the activity Bcl-2 familymember protein can also be readily determined using the assay techniquesdisclosed herein. Furthermore, small organic molecules that can inhibitbinding of HBx to a Bcl-2 family member protein can also be readilyidentified using the methods disclosed herein as well as using acomputer-aided simulation. Synthesis of suitable small organic moleculesis well known to one skilled in the art. Such compounds can be preparedas an array of chemicals and screened for active compounds. In someembodiments, an antibody that can inhibit binding of HBx to a Bcl-2family member protein is used to treat HBV infection. In some instances,the composition of the invention comprises an antibody that can targetBcl-2 homology 3 (BH3)-like motif of hepatitis B virus X protein, Bcl-2family member protein or a combination thereof is used. A suitableantibody for inhibiting binding of HBx to a Bcl-2 family member proteincan be readily prepared by one skilled in the art having read thepresent disclosure using any of the known antibody producing methods.

Yet in other embodiments, the binding inhibitor is capable ofinteracting with Bcl-2 homology 3 (BH3)-like motif of hepatitis B virusX protein. As disclosed herein, it has been discovered by the presentinventors that HBx binds to Bcl-2 and Bcl-xL through its Bcl-2 homology3 (BH3)-like motif. Accordingly, by targeting the BH3-like motif of HBx,or the BH3-binding pocket in Bcl-2 or Bcl-xL, one can inhibit thebinding of HBx to Bcl-2 and/or Bcl-xL.

Still in other embodiments, the composition comprises an expressioninhibitor that is capable of reducing the expression of Bcl-2 familymember protein. Such expression inhibition can be achieved at thetranscription level, translation level, or both. In some particularembodiments, the expression inhibitor is a shRNA that is capable ofreducing the expression of Bcl-2, Bcl-xL, another Bcl-2 family member,or a combination thereof.

Another aspect of the invention provides a method for reducingreplication of hepatitis B virus (HBV) in a cell infected with HBV. Sucha method typically comprises contacting the HBV infected cell with acomposition (i) that is capable of inhibiting binding of hepatitis Bvirus X protein to a Bcl-2 family member protein in said subject; (ii)that is capable of reducing the expression of Bcl-2 family memberprotein in said subject; or (iii) a combination thereof. As discussedabove, suitable binding inhibitors and expression inhibitors can bereadily determined and synthesized by one skilled in the art having readthe present disclosure.

Still another aspect of the invention provides a method for reducing thelevel of cytosolic calcium elevation in a cell, such as hepatocyte,infected with hepatitis B virus (HBV). Such a method generally includescontacting the HBV infected cell with a composition (i) that is capableof inhibiting binding of hepatitis B virus X protein to a Bcl-2 familymember protein in said subject; (ii) that is capable of reducing theexpression of Bcl-2 family member protein in said subject; or (iii) acombination thereof. As discussed above, suitable binding inhibitors andexpression inhibitors can be readily determined and synthesized by oneskilled in the art having read the present disclosure.

Other aspect of the invention includes a method for identifying acomposition that is capable of treating hepatitis B virus (HBV)infection in a mammal. It should be noted that currently there is nosuitable animal model for a relatively rapid molecular genetic analysisof HBV known to one skilled in the art. However, as disclosed herein thepresent inventors have discovered that CED-9 protein of C. eleganscomprises the similar binding domain to HBx as that of Bcl-2 familymember proteins in human. Accordingly, the method for identifying acomposition that is capable of treating HBV infection in a mammalincludes determining the effect of a particular composition in aninteraction between HBV X protein and CED-9 protein of C. elegans. Ingeneral, if a reduction in the interaction between HBV X protein andCED-9 protein in the presence of the composition is observed, then it isan indication that that particular composition is capable of treatingHBV infection in a mammal.

Another aspect of the invention provides a method for treating HBVinfection in a subject by administering a therapeutically effectiveamount of a peptaibol. “Peptaibols” are a large group of shortpolypeptides named after their three characteristic structural features:peptide, Aib (α-aminoisobutyric acid), and C-terminal amino alcohol.They contain a high proportion of unusual amino acids, especially Aib,and are found as secondary metabolites of fungi that have highsimilarity in amino acid sequences among fungi species. Since thediscovery of Alamethicin, the first member of peptaibols, in 1967, todate more than 300 peptaibols have been identified.

Most peptaibols affect biological processes of both microorganisms andanimal cells and cause cell death of human cultured cells. They oftenare inserted into the plasma membrane and oligomerize to formion-permeable channels, leading to sharp voltage changes and death ofthe treated cells. In a large family of peptaibols, Trichokonins (TK)were first isolated and purified from Trichoderma pseudokoningii.Without being bound by any theory, it is believed that like otherpeptaibols, peptaibol TK inserts into lipid membrane and forms anion-channel through oligomer formation. When human tumor cells weretreated with peptaibol TK, these cells showed characteristic apoptosisfeatures, indicating that peptaibol TK promotes apoptotic cell death. Arecent study showed that treatment of hepatocellular carcinoma (HCC)with peptaibol TK caused both apoptosis and autophagic death through acalcium-mediated mechanism. However, the cellular target of peptaibol TKremains unclear.

Programmed cell death or apoptosis is a highly conserved cellularprocess across the animal kingdom. Genetic analysis of programmed celldeath in the nematode C. elegans has been instrumental in identifyingcrucial apoptosis regulators and effectors, leading to theidentification of a conserved cell-killing pathway. Without being boundby any theory, it is believed that in this cell killing pathway, EGL-1,a homolog of the human BH3-only pro-apoptotic proteins, initiatesapoptosis by binding to CED-9, a homolog of the human Bcl-2 cell deathinhibitors, leading to the disassociation of CED-4, a homolog of thehuman apoptotic protease activator Apaf-1, from the inhibitoryCED-4/CED-9 complex tethered on the outer membrane of mitochondria. Thereleased CED-4 then oligomerizes to induce proteolytic activation of theCED-3 zymogen, a homolog of the human caspases, many of which have beenshown to be involved in either apoptosis initiation or execution. Thehighly conserved nature of the apoptosis pathway is demonstrated by thefindings that human Bcl-2 protein can partially substitute for thefunction of CED-9 in C. elegans and that a human viral protein, thehepatitis B virus X protein (HBx) containing a BH3-like motif, caninduce both apoptosis and necrosis in both C. elegans and humanhepatocytes by directly targeting CED-9 and Bcl-2 family proteins.

Recently, C. elegans has emerged as a powerful animal model for drugscreens and subsequent target identification. For example, studies in C.elegans reveal that the Benzenoid chemicals, including naphthalene andpara-dichlorobenzene, target caspases in both C. elegans and humans toinhibit apoptosis, resulting in cell death abnormality in worms andtumorigenesis in human.

Some aspects of the invention are based on the discovery by the presentinventors that peptaibol TK promotes apoptosis in C. elegans. Withoutbeing bound by any theory, it is believed that peptaibol TK promotesapoptosis by targeting the cell death inhibitor CED-9, thus leading toincreased CED-4 release from the inhibitory CED-9/CED-4 complex incooperation with EGL-1. Other aspects of the invention are based on thediscovery by the present inventors that peptaibol TK enhancesHBx-induced cell death in C. elegans, in HBV transgenic mice, and inhuman hepatocytes. These results show that peptaibol TK is a potentialanti-tumor drug that can also be used to treat HBV infection. In someembodiments, peptaibol TK comprises peptaibol TK VI (SEQ ID:NO 1),peptaibol TK VII (SEQ ID:NO 2) or peptaibol TK VIII (SEQ ID:NO 3).

It should be appreciated that the scope of the invention also includes aderivative or a modified peptaibols. As used herein, a “derivative” ofpeptaibol refers to a modified peptaibol in which one or more amino acidin the peptide of peptaibol of interest is replaced with a functionallysimilar amino acid. For example, naturally occurring residues aredivided into groups based on common side chain properties:

(1) hydrophobic: norLeucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr;

(3) acidic: Asp, Glu;

(4) basic: Asn, Gln, His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro; and

(6) aromatic: Trp, Tyr, Phe.

Thus, in general, a modified peptaibol refers to a peptaibol in whichone or more, typically five or less, often three or less and more oftentwo or less amino acids of a peptaibol of interest have been replacedwith an amino acid having a similar side chain property. For example, “aderivative” of peptaibol TK refers to a homolog, an analog of peptaibolTK. Exemplary derivatives of peptaibol TK include, but are not limitedto:

-   -   (i) a peptaibol whose peptide fragment is derived from peptaibol        TK that contains α-aminoisobutyric acid and an alcohol and have        a similar activity (i.e., in vitro assay activity within ±25%,        typically within ±10%, and often within ±5% activity;    -   (ii) peptaibol in which one or several amino acids of the        natural peptaibol TK sequence have been substituted by other        amino acids;    -   (iii) peptaibol modified at the N- and/or C-terminal end of the        peptide sequence of peptaibol TK, for example, by substitution;        thus, esters and amides can be considered as derivatives;    -   (iv) peptaibol TK peptides the modification of which prevents        their destruction by proteases or peptidases, as well as to        peptide-PEG-conjugates derived from the basic sequence of        peptaibol or its fragment;    -   (v) modified peptides of peptaibol TK which are derived from the        chain of peptaibol TK peptide or its fragment and wherein one or        several of the amino acids of the sequence have been substituted        by genetically encoded or not genetically encoded amino acids or        peptidomimetics;    -   (vi) peptides of peptaibol TK having conservative substitutions        of amino acids as compared to the natural sequence of peptaibol        TK in one or several positions. A conservative substitution is        defined as the side chain of the respective amino acid being        replaced by a side chain of similar chemical structure and        polarity, the side chain being derived from a genetically coded        or not genetically coded amino acid. Families of amino acids of        this kind having similar side chains are known in the art. They        comprise for instance amino acids having basic side chains        (lysine, arginine, histidine), acidic side chains (aspartic        acid, glutamic acid), uncharged polar side chains (glycine,        aspartamic acid, glutamine, serine, threonine, tyrosine,        cysteine), non-polar side chains (alanine, valine, leucine,        isoleucine, proline, phenylalanine, methionine, tryptophan),        β-branched side chains (threonine, valine, isoleucine) and        aromatic side chains (tyrosine, phenylalanine, tryptophane,        histidine). Such conservative substitutions of side chains are        typically carried out in non-essential positions. In this        context, an essential position in the sequence is one wherein        the side chain of the relevant amino acid is of significance for        its biological effect.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting. Inthe Examples, procedures that are constructively reduced to practice aredescribed in the present tense, and procedures that have been carriedout in the laboratory are set forth in the past tense.

EXAMPLES Example 1

Immunoprecipitation assays. HepG2 cells transfected with thepcDNA3.1-Flag-HBx constructs or the pHBV replicons (wild-type andG124L/I127A mutations) were lysed and precipitated using an anti-Flagantibody or an anti-HBx antibody. The proteins pulled down with HBx weredetected by either immunoblotting analysis or mass-spectrum analysis.

Calcium imaging and analysis. HepG2 cells co-transfected withpcDNA3-mCherry and pcDNA3.1-Flag-HBx constructs (wild-type orG124L/I127A mutations) were incubated for 35 min with 4 μM Fura-2-AM and0.04% Pluronic solution 48 hr post transfection, washed three times withbuffer, and incubated for an additional 15 min to allow for cleavage ofthe acetoxymethyl (AM) ester, which trapped Fura-2 in the cells. Datawere collected using the Metafluor software and analyzed by Excel.Statistical analysis was performed using t-test in KaleidaGraph program.The error bars indicate SEM (standard error of the mean).

Quantification of HBV DNA replication and HBcAg. Southern hybridizationanalysis and quantitative real-time PCR were used to quantify the amountof HBV replication DNA intermediates isolated from HepG2 cells or frommouse livers. The level of cytoplasmic HBcAg was measured bychemiluminescence using a commercial assay kit.

Hydrodynamic Injection. 30 μg of the pHBV replicon and 3 μg ofpcDNA3-GFP were injected into the tail veins of Balb/c mice within 5seconds in a volume of PBS equivalent to 10% of the mouse body weight.Livers of the injected mice were assayed for HBcAg and viral DNA twodays after injection.

Molecular Biology. The HBx cDNA clone was obtained from Dr. Xiao-KunZhang (The Burnham Institute for Medical Research). Standard methods ofcloning, sequencing, and polymerase chain reaction amplification (PCR)were employed. To make HBx mammalian expression constructs, a DNAfragment encoding Flag-HBx was amplified by PCR and subcloned into thepcDNA3.1(+) vector via its Nhe I and EcoR V sites. The HBx mutantconstructs containing G124L and I127A substitutions were generated usinga QuickChange Site-Directed Mutagenesis kit (Stratagene Inc.) andconfirmed by DNA sequencing. The pHBV replicon contains a 140% DNA copyof the HBV genome and replicates in an HBx-dependent manner in HepG2cells. pHBV containing HBx(G124L, I127A) was made by Quick-Changesite-directed mutagenesis and confirmed by DNA sequencing.

Cell Culture. Human HepG2 cells were grown in DMEM with 10% fetal bovineserum (FBS; Sigma-Aldrich). Transfection of HepG2 cells was carried outusing Effectene Transfection Reagent (Qiagen) following themanufacturer's protocol. A transfection efficiency of 15 to 30% wasroutinely achieved. All transfection experiments were performed 24 hoursafter plating. 1 μg of pcDNA3.1, pcDNA3.1-Flag-HBx, orpcDNA3.1-Flag-HBx(G124L, I127A) was diluted in 100 μl of theDNA-condensation buffer (Buffer EC) supplied by the manufacturer(Qiagen). 8 μl of enhancer and 10 μl of Effectene were sequentiallyadded to the mixture, each followed by vortexing and incubation at roomtemperature per manufacture protocol. After that, the mixture wassupplemented with 0.6 ml complete medium and added to cells.Co-transfection of an enhanced GFP (EGFP)-expressing plasmid pEFGP-C1 (1μg) was included, where appropriate, to monitor transfection efficiencywhen performing flow cytometry analysis in the cell killing assays.

Co-Immunoprecipitation Assays. Co-immunoprecipitation experiments wereperformed using an antibody (M2) to the Flag epitope (Sigma) or ananti-HBx antibody (16F9). Briefly, HepG2 cells transfected withpcDNA3.1-Flag-HBx or pHBV constructs (wild-type or mutant) were lysed inlysis buffer (100 mM NaCl, 0.5 mM MgCl₂, 0.15 mM CaCl₂, 1% (v/v) NP-40,10 mM Tris-HCl, pH 8.0) containing protease inhibitor cocktail tablets(Roche). Cell debris was removed by centrifugation at 10,000 g for 10minutes at 4° C. The cell lysate was precleared with Protein G Sepharosebeads (GE healthcare) and subsequently incubated with the M2 or 16F9antibody for 1 hour with gentle shaking at 4° C. Protein G Sepharosebeads were then added and the incubation continued for another 2 hours.The beads were washed five times with the lysis buffer. The boundproteins were resolved on a 15% SDS polyacrylamide gel and detected byimmunoblotting using anti-Bcl-2, anti-Bcl-xL, and anti-Mcl-1 antibodies(Cell Signaling Technology), respectively.

Flow Cytometry. 36 hours post-transfection, living and dead HepG2 cellswere scraped into the cell growth medium and precipitated bycentrifugation. Cells from one well of a six-well plate (˜6×10⁵ cells)were washed in cold PBS twice and resuspended in 600 μl AnnexinV-binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl₂, pH 7.4).Labeling of cells by Propidium Iodide (Sigma) and Annexin-V pacific blue(Invitrogen) was carried out according to the protocol provided byInvitrogen. Briefly, 100 μl of the suspended cells were transferred to anew tube. 5 μl of Annexin-V Pacific Blue (Invitrogen) and 5 μl ofPropidium Iodide (Sigma) were added to each tube and incubated for 30minutes at room temperature in the dark. 400 μl of Annexin V-bindingbuffer were then added to each tube. Cells were analyzed on the CyAn™ADP Analyzer (DakoCytomation) with background gates set to excludenon-transfected GFP(−) cells and to restrict Annexin-V Pacific Bluestaining alone, or Propidium Iodide staining alone, to fewer than 0.5%positive events. Data were collected from more than 10,000 cells foreach sample.

Cytosolic Calcium Measurement. HepG2 cells were co-transfected withpcDNA3-mcherry and pcDNA3.1-Flag-HBx constructs (wild-type or mutant)using lipofectamine LTX (Invitrogen). After 48 hours or later, cellswere washed with the HHBSS buffer (Hank's Balanced Salt Solutionsupplemented with 20 mM HEPES and 11 mM Glucose) three times and thenincubated with 4 μM Fura-2-AM and 0.04% Pluronic solution for 35minutes. Cells were rinsed with HHBSS three times and left with HHBSSfor another 15 minutes to allow for cleavage of the acetoxymethyl (AM)ester, trapping Fura-2 inside the cells.

Imaging experiments were performed on an Axiovert 200M invertedfluorescence microscope (Zeiss) with a Cascade 512B CCD camera (RoperScientific), equipped with a Xenon Arc lamp (XBO75), and 340/26 and380/10 excitation filters, a 450 nm dichroic mirror, and a 535/45emission filter. Excitation and emission filters were placed in filterwheels external to the microscope controlled by a Lambda 10-3 filterchanger (Sutter Instruments) to allow for rapid acquisition of ratioimages. Images were collected using Metafluor software (UniversalImaging). All images were collected on healthy cells with similarmCherry intensity using a 40× oil objective and were backgroundcorrected by generating a region of interest (ROI) on a blank area ofthe coverslip and subtracting the fluorescence intensity of eachexcitation channel. The background corrected intensities at 340 nM and380 nM excitation were used to calculate the Fura-2 340/380 ratio.

To determine the resting [Ca²⁺] in the cytosol, cells were treated with5 μM ionomycin and 5 mM EGTA in Ca²⁺-free HBSS to obtain the ratio ofthe unbound indicator (R_(min)). Cells were then washed with Ca²⁺-freeHBSS for three times and treated with 5 μM ionomycin and 10 mM Ca²⁺ inHBSS to obtain the ratio of the calcium-saturated indicator (R_(max)).Ca²⁺ concentrations were calculated using the reported K_(d) value ofFura-2 and the experimentally derived R, R_(min), R_(max), S_(f) andS_(b) (the emission intensity at 380 nm for Ca²⁺-free and Ca²⁺-boundFura-2, respectively) in each individual cell according to the followingequation:

$\lbrack {Ca}^{2 +} \rbrack = {K_{d} \times \frac{( {R - R_{\min}} )}{( {R_{\max} - R} )} \times {\frac{S_{f}}{S_{b}}.}}$Details on the microscope, sensor calibration, and conversion of Fura-2ratios into Ca²⁺ concentrations have been described in J Biol Chem.,1985, 260, 3440-3450. The results were presented as the fold-increase ofthe calcium concentration in cells transfected with pcDNA3-Flag-HBx overthat in cells transfected with the empty vector.

Southern Blot Analysis. HepG2 cells (3×10 cm plate per sample)transfected with the pHBV replicon (wild-type or mutant) were washedtwice with cold phosphate-buffered saline (PBS) and lysed in 750 μl NETbuffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 100 mM NaCl, 0.5% NonidetP-40) per 10 cm plate for 1 hour. HepG2 cell lysates were clarified bycentrifugation at 12000 g for 30 minutes at 4° C. The supernatant wasadjusted to 6 mM CaCl₂ and treated with 100 μg/ml of Micrococcalnuclease for 30 minutes at 37° C. The reaction was stopped by additionof EDTA to a final concentration of 25 mM. Proteins were digested with0.2 mg/ml proteinase K and 0.5% SDS for overnight at 37° C. Nucleicacids were purified by phenol/chloroform extraction and ethanolprecipitation. After centrifugation at 12000 g for 30 minutes at 4° C.,the pellet was resuspended in 10 μl TE buffer. DNA samples were resolvedon a 1.2% agarose gel, transferred to nylon membrane (Bio-Rad), andhybridized with a digoxin-labeled DNA fragment covering the entire HBxgene.

Northern Blot Analysis. Total cellular RNA was extracted fromtransfected cells using the Trizol reagent (Invitrogen) according to themanufacturer's instructions. 20 μg of RNA were separated on 1%denaturing formaldehyde agarose gel, transferred onto nylon membranes,and hybridized to probes covering the whole HBV genome, prepared asdescribed above.

Quantitative Real-Time PCR Analysis. Intracellular HBV replicatingintermediates were isolated from cytoplasmic viral core particles asdescribed in EMBO J., 1999, 18, 5019-5027. The pHBV plasmid was used asthe standard for quantification and serial dilutions from 2×10⁸ to 200IU/ml were prepared. The primer sequences for quantitative PCR werechosen carefully from regions coding for HBV S antigen and Polymerase.Sequences of primer Set 1 (corresponding to HBS): forward primer:5′-GTGTCTGCGGCGTTTTATCA-3′ (SEQ ID:NO 4), reverse primer:5′-GACAAACGGGCAACATACCTT-3′ (SEQ ID:NO 5). Primer Set 2 (correspondingto Polymerase): forward primer: 5′-TACTAGTGCCATTTGT-TCAGTGG-3′ (SEQID:NO 6), reverse primer: 5′-CACGATGCTGTACAGACTTGG-3′ (SEQ ID:NO 7).Real-time PCR analyses were performed using a SYBR Green Premix Ex TaqKit (Takara Bio) in an ABI Prism 7500 PCR system (Applied Biosystems).PCR products were analyzed by fluorescence. Each standard dilution wassubjected to 2 PCR runs in at least two independent experiments. Basedon the mean threshold cycles (C_(T)) for each dilution, a linearregression was carried out with the C_(T) values as a function of thedecadic logarithm of the number of template molecules per reaction.Least-squares regression analysis, performed by the ABI prism 7500,plotted C_(T) as a function of nominal input number. The measured rawcopy number from each reaction was calculated using the C_(T) value ofeach PCR interpolated against the linear regression of the standardcurve. Each DNA specimen was subjected to 4 PCR runs with at least threeindependent experiments.

HBV Core Antigen Analysis. The amounts of HBcAg were determined bychemiluminescence using a commercial assay kit (Wantai, Beijing, China).

Mice. Balb/c mice (male, 6 to 7 weeks old) were divided into two groups(6 mice for each injection group). 30 μg of the pHBV replicon (wild-typeor mutant) plus 3 μg of pcDNA3-GFP were injected into the tail veins ofmice within 5 seconds in a volume of PBS equivalent to 10% of the mousebody weight. Livers of these mice were collected and assayed for thelevels of HBcAg and viral DNA, respectively, two days after injection asdescribed in Proc Natl Acad Sci USA., 2006, 103, 17862-17867.

shRNA-Mediated Knockdown in HepG2 Cells. Validated Mission® shRNAlentiviral particles targeting human Bcl-2 or the control shRNAlentiviral particles were obtained from the Functional Genomic Facilityof the University of Colorado. A short hairpin RNA targeting humanBcl-xL was produced using the lentivirus-based shRNA delivery vectorpLL3.7-neo. Viral particles targeting Bcl-xL were produced in HEK 293FTcells and used to infect HepG2 cells. For shRNA knockdown experiments,cells were plated 24 hours before infection. Lentiviral particles wereadded in the presence of hexadimethrine bromide at the recommendedmultiplicity of infection. Infected cells were selected in media withpuromycin (1 μg/mL; Sigma-Aldrich) or neomycin (0.5 mg/mL;Sigma-Aldrich). The Bcl-2 mRNA sequence targeted by the shRNA is:5′-CCGGGAGATAGTGATGAAGTA-3′ (SEQ ID:NO 8). The Bcl-xL mRNA sequencetargeted by the shRNA is: 5′-GTGGAACTCTATGGGAACAAT-3′ (SEQ ID:NO 9).

Results

Analyses of HBx activities in C. elegans by the present inventors showedthat HBx interacts with CED-9 in C. elegans to induce cytosolic calciumincrease and cell death. Moreover, HBx also interacted with human Bcl-2and Bcl-xL in vitro through its BH3-like motif and this interaction wasdisrupted by two substitutions in conserved residues of the BH3-likemotif (G124L and I127A). The present inventors also conductedexperiments to determine whether HBx interacts with Bcl-2 and/or Bcl-xLin human hepatic HepG2 cells by the co-immunoprecipitation assay. InHepG2 cells transfected with pcDNA3.1-Flag-HBx, endogenous Bcl-2 andBcl-xL, but not the anti-apoptotic Bcl-2 family member Mcl-1, wereco-precipitated with Flag-HBx using an antibody to the Flag epitope. Incontrast, no Bcl-xL and only a trace amount of Bcl-2 wereco-precipitated with Flag-HBx(G124L, I127A), which was expressed inHepG2 cells at a comparable level to Flag-HBx. These results indicatethat HBx associates with Bcl-2 and Bcl-xL in human cells through itsBH3-like motif. Importantly, in HepG2 cells transfected with a 140%head-to-tail DNA copy of the HBV genome (pHBV) with or withoutHBx(G124L, I127A) mutations, endogenous Bcl-2 and Bcl-xL, but not Mcl-1,were co-precipitated with HBx, but not with HBx(G124L, I127A), using anantibody to HBx. Therefore, HBx expressed from its native promoter in areplicating HBV genome associates with endogenous Bcl-2 and Bcl-xL inhuman hepatic cells through its BH3-like motif.

The cell-killing activity of HBx in human cells was analyzed by stainingHBx-transfected HepG2 cells with Annexin-V Pacific Blue and propidiumiodide (PI) to distinguish living cells from apoptotic and necroticcells. Flow cytometry analysis of cells transfected withpcDNA3.1-Flag-HBx showed 10.6% apoptotic cells (Annexin-V positive andPI negative) and 7.9% necrotic cells (Annexin-V positive and PIpositive). Significantly less cell death was observed in HepG2 cellstransfected with the same amount of pcDNA3.1-Flag-HBx(G124L, I127A)(4.87% apoptotic cells and 1.14% necrotic cells) or empty pcDNA3.1vector (1.14% apoptotic cells and 0.32% necrotic cells). Therefore, asin C. elegans, HBx utilizes its BH3-like motif to bind anti-apoptoticBcl-2 and Bcl-xL proteins and induces both apoptosis and necrosis inhuman hepatocytes.

Since calcium signaling is an important event downstream of HBx, whetherthe BH3-like motif of HBx is necessary for HBx-induced elevation ofcytosolic calcium was examined. Cytosolic calcium in HepG2 cellstransfected with pcDNA3.1-Flag-HBx or pcDNA3.1-Flag-HBx(G124L, I127A)was determined using the ratiometric fluorescent calcium indicatorFura-2. It was found that the resting calcium concentration wassignificantly increased upon expression of HBx compared to thevector-only control, whereas expression of HBx(G124L, I127A) failed todo so. It is believed that the HBx-induced calcium elevation was not dueto increased cell death, because a similar level of calcium increase wasobserved in the presence of Z-VAD, a pan-caspase inhibitor that blockscell death. These results indicate that HBx can induce an increase incytosolic calcium that is dependent on its BH3-like motif andassociation with Bcl-2 family proteins.

Given the role of HBx in HBV DNA replication, whether interactionsbetween HBx and Bcl-2 proteins are important for HBV replication wasexamined. Cytoplasmic viral core particles, where HBV DNA replicationoccurs, were isolated from HepG2 cells transfected with the pHBVreplicon with or without the HBx(G124L, I127A) mutations. The level ofHBV DNA replication was examined by southern blot analysis. Comparedwith cells transfected with wild-type pHBV, HBV DNA replication wassignificantly reduced in cells transfected with the mutant pHBV. Thelevel of the HBV core protein (HBcAg), which correlates with the levelof HBV DNA, was also greatly reduced in cells transfected with themutant HBV genome. Northern blot analysis showed no reduction in HBVpregenomic (pg)/precore (pc) RNA, preS/S mRNA, or HBx mRNA in cellstransfected with the mutant pHBV replicon. Quantitative real-timepolymerase chain reaction (Q-PCR) analysis of isolated viral particlesrevealed a 8-9 fold reduction in HBV DNA replication in cellstransfected with the mutant HBV genome compared with cells transfectedwith the wild-type HBV genome. These results indicate that the BH3-likemotif of HBx is critical for HBV DNA replication but dispensable for HBVtranscription. Interestingly, HBV DNA replication in cells transfectedwith the mutant HBV genome was largely rescued by treatment with 5 μMionomycin, an ionophore that increases cytosolic calcium. This resultshows that increased cytosolic calcium is an important signaling eventdownstream of HBx interaction with Bcl-2 proteins that stimulates HBVDNA replication.

The role of HBx interaction with Bcl-2 proteins for HBV replication wasexamined in an established mouse model of chronic HBV infection. ThepHBV replicon with wild-type HBx or HBx(G124L, I127A) was introducedinto BALB/C mice (n=6) through hydrodynamic tail vein injection, alongwith a pcDNA3-GFP reporter as a control for injection efficiency.Cytoplasmic viral core particles were isolated from the liver 2 daysafter injection and subjected to southern blot analysis. The averagelevel of replicative DNA intermediates in livers of mice receiving themutant pHBV replicon was reduced by 2-3 fold compared to that of micereceiving the wild-type pHBV replicon. Expression of intrahepatic HBVcore antigen (HBcAg) was similarly reduced in mice receiving the mutantpHBV replicon. These results confirm the role of the BH3-like motif ofHBx, and thus the association of HBx with Bcl-2 family proteins, for HBVDNA replication in HBV-infected liver.

The role of Bcl-2 proteins for HBV replication was determined byknocking down the expression of Bcl-2 or Bcl-xL in HepG2 cells throughRNA interference (RNAi). Compared with control short hairpin RNA(shRNA), Bcl-2 and Bcl-xL shRNA significantly reduced the expression ofBcl-2 and Bcl-xL in HepG2 cells. Q-PCR analysis of isolated viralparticles revealed a 21-41% reduction in HBV DNA replication in cellsinfected by lentivirus expressing Bcl-2 or Bcl-xL shRNA, compared tocells with control shRNA. Overexpression of the anti-apoptotic Mcl-1protein, which does not interact with HBx, in cells treated with Bcl-2or Bcl-xL shRNA did not significantly prevent reduction of HBV DNAreplication, indicating that decreased HBV DNA replication caused byloss of Bcl-2 or Bcl-xL is unlikely due to impaired survival of the hostcells. Moreover, RNAi knockdown of Bcl-2 or Bcl-xL dampened but did notcompletely eliminate intracellular calcium increase induced by HBx,which is consistent with the finding that Bcl-2 or Bcl-xL knockdownreduced but did not block HBV DNA replication and indicates that Bcl-2and Bcl-xL are partially redundant in mediating HBx functions. Bcl-2 andBcl-xL double knockdown cells were not viable for analysis of HBV viralreplication and intracellular calcium changes. These results indicatethat Bcl-2 and Bcl-xL are important for HBV viral replication, andtogether with the findings described above, provide strong evidence thatHBx targets both Bcl-2 and Bcl-xL to increase intracellular calcium andto promote HBV DNA replication.

Discussion

Despite the role of HBx in HBV pathogenesis and oncogenesis,identification of HBx host targets has remained a major challenge in thelast three decades. The intricacy of HBx activities, the lack of asuitable animal model to study HBV infection, variability among cellculture assays, and the complexity of the mammalian genome, whichencodes at least six Bcl-2 family proteins, have all contributed to thelongstanding questions regarding the functions of HBx, its interactionswith host targets, and its mechanisms of action. The present inventorshave engineered a C. elegans animal model to identify HBx targets anddownstream signaling pathways (see Example 2). Mimicking the initialcellular events that unfold following liver infection by HBV, HBxinduced both apoptosis and necrosis in C. elegans through canonical celldeath pathways. Interestingly, a unique gain-of-function mutation(G169E) in the Bcl-2 homolog CED-9, which inhibits cell death in C.elegans by blocking the binding of the endogenous BH3-only cell deathinducer EGL-1 to CED-9, also completely blocked the interaction betweenCED-9 and the BH3-like motif of HBx and HBx-induced cell death in C.elegans. Remarkably, Bcl-2 can fully substitute for CED-9 in C. elegansto mediate HBx-induced cell killing, indicating that Bcl-2 likelyinteracts with HBx in mammals. The present inventors have demonstratedhere that HBx associates with Bcl-2 and Bcl-xL in human hepatocytesthrough its BH3-like motif and that this protein interaction is crucialfor HBx-induced cytosolic calcium elevation, cell death, and viral DNAreplication. These findings indicate that molecular mimicry ofendogenous BH3-only proteins by HBx enables its interactions withconserved host targets and hijacking of cell signaling pathways tobenefit viral infection.

Calcium signaling is a critical event downstream of HBx expression thatpromotes inter alia HBV replication, core assembly, cell death, andother HBx functions. HBx has been hypothesized to effect mitochondriapermeability transition (MPT), which is important for intracellularcalcium homeostasis and cell death. Importantly, both Bcl-2 and Bcl-xLare mitochondrial proteins and have been implicated in regulating MPT.HBx binding to Bcl-2 and Bcl-xL is critical for calcium regulation byHBx, since expression of HBx, but not HBx(G124L, I127A), which fails tobind Bcl-2 and Bcl-xL, triggered elevation of cytosolic calcium inhepatocytes. The finding that G124L/I127A mutations in the BH3-likemotif of HBx greatly reduced HBV DNA replication in human and mousehepatocytes, which can be substantially rescued by restoring cytosoliccalcium with ionomycin, and the observation that RNAi knockdown ofeither Bcl-2 or Bcl-xL significantly compromised HBx-inducedintracellular calcium increase and HBV replication, provided furtherconfirmation that HBx targets Bcl-2 proteins to trigger cytosoliccalcium elevation required for HBV replication and other events such ascell death.

Hepatocarcinogenesis is a complex and poorly understood process. Chronichepatocyte cell death induced by HBV infection or carcinogens maytrigger cycles of inflammation, immune response, compensatory tissueregeneration, and the acquisition of oncogenic mutations that lead todevelopment of HCC. On the other hand, hepatocyte expression ofpro-survival factors, such as Bcl-2 and p38α kinase, has been shown tobe effective in preventing HCC development. Moreover, HBV viralreplication plays an important contributing role inhepatocarcinogenesis. The development and progression of HCC in chronicHBV patients strongly correlates to the viral DNA level in adose-dependent manner. Therefore, blocking HBV viral replication andHBV-induced cell death represents an effective strategy to treat chronicHBV patients and to prevent the development of HCC. The presentinventors have discovered that the BH3-like motif of HBx is necessaryfor HBx binding to Bcl-2 family proteins, which results in elevatedcytosolic calcium, efficient viral replication, and HBV-induced celldeath. Therefore, therapeutically targeting the BH3-like motif of HBx orthe interactions between HBx and Bcl-2 proteins is a new and effectivestrategy to treat chronic HBV patients and to prevent development ofHCC.

Example 2

This example shows inter alia that expression of HBx in C. elegansinduces both necrotic and apoptotic cell death, mimicking an early eventof liver infection by HBV. Genetic and biochemical analyses indicatethat HBx interacts directly with the Bcl-2 homolog CED-9 through a Bcl-2homology 3 (BH3)-like motif to trigger both cytosolic Ca²⁺ increase andcell death. Importantly, Bcl-2 can substitute for CED-9 in mediatingHBx-induced cell killing in C. elegans, suggesting that CED-9 and Bcl-2are conserved cellular targets of HBx. A genetic suppressor screen ofHBx-induced cell death has produced many mutations, including mutationsin key regulators from both apoptosis and necrosis pathways, indicatingthat this screen can identify new apoptosis and necrosis genes. Thepresent inventors have found that C. elegans serve as an animal modelfor identifying crucial host factors and signaling pathways of HBx andcan be used in development of strategies to treat HBV-induced liverdisorders.

C. elegans strains and cell death assays. Animals were grown andmaintained using standard protocols. Germline transformation was used togenerate transgenic C. elegans strains expressing HBx and other genes.Embryonic lethality was scored 24 hours after heat-shock treatment. PLMkilling by HBx was scored as described in Nat Struct Mol Biol., 2008,15, 1094-1101.

GST pull-down assays. GST-HBx proteins were purified using GlutathioneSepharose beads and binding to CED-9 or Bcl-2 family proteins wasassayed as described in Nat Struct Mol Biol., 2008, 15, 1094-1101.

Calcium imaging and analysis. A C. elegans strain with an integratedP_(mec-4)YC2.12 cameleon transgene (bzIs17) was used to quantifyrelative cytosolic calcium levels in PLM neurons by the FRET microscopy.

Worm Strains and Culture Conditions. C. elegans strains were cultured at20° C. using standard procedures. The N2 Bristol strain was used as thewild-type strain. The following alleles were used in the geneticanalyses: LGI, mec-6(e1342); LGIII, ced-4(n1162), clp-1(tm690),cnx-1(nr2009), ced-9(n1950, n2812); LGIV, itr-1(sa73), ced-3(n2433);LGV, crt-1(bz29), unc-68(e540), egl-1(n3082), cyn-1(tm4171); LGX,asp-3(tm4559), asp-4(ok2693). Detailed allele information is describedin Wormbase (www.wormbase.org/). In addition to these strains, bzIs8 isan integrated transgene located on LGX and contains a P_(mec-4)GFPconstruct, which directs GFP expression in six C. elegans touch receptorneurons. smIs98 is an integrated transgene located on LGII and containsP_(mec-3)GFP and P_(mec-7)HBx constructs. smIs3 is an integratedtransgene containing P_(mec-3)GFP. bzIs17 is an integrated transgenecontaining P_(mec-4)YC2.12, which directs expression of the YC2.12cameleon calcium sensor under the control of the mec-4 gene promoter.smIs451 is an integrated transgene containing only P_(mec-7)HBx. Allintegrated transgenes were backcrossed six times with the N2 strainbefore being used for further genetic analyses.

Embryonic Lethality Assay. Gravid transgenic adults were placed onplates for 2 hr at 20° C. to let them lay eggs. The plates were thenheat-shocked at 33° C. for 1 hr and returned to 20° C. for 1 hr beforeremoving all adult worms. After 24 hr at 20° C., the transgenic embryoswere scored for embryonic lethality.

EMS Mutagenesis. EMS mutagenesis was carried out. Briefly, smIs98;smEx[P_(hsp)HBx+P_(hsp)GFP] L4 larvae were exposed to 47 mM EMS for 4 hrwith agitation. The F1 progeny of mutagenized animals were cloned out,and the F2 embryos were subjected to heat-shock treatment at 33° C. for1 hr and returned to 20° C. for 1 hr. This heat-shock treatment wasrepeated 3 more times to ensure 100% killing of embryos by HBx. Thesurviving larvae with robust GFP expression in many cells wereidentified as putative suppressor mutants, which were subjected to thesecondary screen using smIs98. Only those mutants in which HBx-inducedtouch cell death was completely or partially suppressed were consideredas true suppressors and analyzed further.

Quantification of PLM Killings by HBx. PLM neurons were scored in L4larvae in the presence of the integrated transgene, smIs98 or bzIs8,using a Nomarski microscope equipped with epifluorescence.

Transgenic Worms. Germline transformation was performed as described inCell Death Differ., 2009, 16, 1385-1394. The P_(hsp)HBx constructs (at25 ng/μl each) were injected into animals with the appropriate geneticbackground using P_(sur-5)GFP (25 ng/μl) as a co-injection marker, whichdirects GFP expression in all somatic cells in most developmentalstages. The P_(mec-7)HBx construct or its mutant derivatives (25 ng/μl)was injected into bzIs8; unc-76(e911) animals using p76-16B (5 ng/μl) asa co-injection marker, which rescues the Uncoordinated defect of theunc-76(e911) mutant. The P_(mec-7)CED-9, P_(mec-7)CED-9ΔTM, orP_(mec-7)hBcl-2 construct (25 ng/μl) was injected into smIs98;ced-9(n1950) animals using pRF4 (50 ng/μl) as a co-injection marker,which causes a Roller phenotype in transgenic animals.

Molecular Biology. Standard methods of cloning, sequencing, andpolymerase chain reaction amplification (PCR) were employed. Briefly,full-length HBx cDNA was subcloned into the pGEX-4T-2 vector via itsEcoR I and Not I sites to generate the pGEX-4T-2-HBx protein expressionvector. To make P_(hsp)HBx constructs, full-length HBx cDNA wassubcloned into C. elegans heat shock vectors, pPD49.78 and pPD49.83, viaNhe I and Kpn I sites. P_(mec-7)HBx was constructed by subcloning HBxcDNA into the pPD52.102 vector via its Nhe I and Kpn I sites. The HBxmutant constructs containing G124L, I127A, E125A or E126A substitutionswere generated using a QuickChange Site-Directed Mutagenesis kit(Stratagene Inc.) and confirmed by DNA sequencing. To make Bcl-2, Bcl-xLand Bax protein expression vectors, the coding regions for humanBcl-2(1-218), human Bcl-xL(1-209), and mouse Bax(1-173) were amplifiedby PCR and subcloned into the pET-19b vector via its Nde I and Xho Isites, respectively. pET-24a-CED-9(68-251) and pET-24a-CED-9(68-251;G169E) were generated by Parrish et al. (7). P_(mec-7)CED-9 andP_(mec-7)hBcl-2 were generated by subcloning the full-length ced-9 andhuman Bcl-2 cDNA fragments into the pPD52.102 vector via its Nhe I andEcoR V sites, respectively. P_(mec-7)CED-9ΔTM was generated bysubcloning a cDNA fragment encoding CED-9 amino acids 1-251 into thepPD52.102 vector via its Kpn I and EcoR V sites.

Protein Expression and Purification. GST-HBx proteins (wild-type ormutants) were expressed in Escherichia coli strain BL21(DE3). Thesoluble fraction of the E. coli lysate was incubated with GlutathioneSepharose beads (Pharmacia) and purified GST-HBx proteins were elutedwith 10 mM reduced glutathione (Amersham). CED-9(68-251), humanBcl-2(1-218), human Bcl-xL(1-209), and mouse Bax(1-173) proteins wereexpressed individually in BL21(DE3) as either a C-terminally orN-terminally six histidine-tagged protein using a pET-24a or pET-19bvector (Novagen), respectively. They were affinity purified using TalonMetal Affinity Column (Clontech) and eluted with 250 mM imidazole.

GST Fusion Protein Pull-Down Assays. Purified GST-HBx proteins or GSTprotein (2.5 μg each) immobilized on Glutathione Sepharose beads(Pharmacia) were incubated with 5 μg of purified CED-9(68-251)-His₆,CED-9(68-251; G169E)-His₆, His₆-hBcl-2(1-218), His₆-hBcl-xL(1-209), orHis₆-mBax(1-173) in a binding buffer containing 25 mM Tris-HCl (pH 7.5),150 mM NaCl, 0.1% Nonidet P40, 10% glycerol, 1 mM phenylmethylsulphonylfluoride, and 5 mM dithiothreitol at 4° C. for 2 hr. One portion of theincubation mix was analyzed by western blot to examine the input levelsof GST-HBx proteins and Bcl-2 family proteins using an antibody to GST(B-14) or the six-histidine tag (H15), respectively (Santa CruzBiotechnology, Inc.). The Sepharose beads were then washed five timeswith the same buffer before the bound proteins were resolved on a 15%SDS polyacrylamide gel and detected by immunoblotting.

Structural Modeling. Homology modeling of the complex structure betweenthe HBx Peptide (residues 120-128) and CED-9 was performed using thepublished complex structure between the EGL-1 BH3 domain and CED-9 as atemplate (PDB code: 1TY4). The model was further optimized using theprogram COOT manually to be reasonable. Both modeling figures werecreated by the PyMOL program and labeled using Adobe Illustrator CS4.

Chemical Treatment of C. elegans. Worms were treated with chemicalsusing an oil-base protocol. Briefly, Thapsigargin (Sigma) or CyclosporinA (Sigma) was dissolved in DMSO and then diluted it in 100% soybean oil(Crisco). L4 larval stage hermaphrodite animals were placed ontostandard NGM plates seeded with OP50. Oil solutions containing thechemicals were spread (0.8-1.0 ml) onto each plate so that the NGMsurface was completely covered by oil. Worms lived at the interface ofthe NGM medium and the oil. F1 progeny was examined for PLM cell deathor the FRET ratio in PLMs.

Calcium Imaging and Analysis in C. elegans. A C. elegans strain with anintegrated P_(mec-4)YC2.12 cameleon transgene (bzIs17) was used toquantify relative cytosolic calcium levels in PLM neurons. Animals atthe L1 larval stage were immobilized on a 2% agar pad in a solutioncontaining 0.3 M 2,3-butanedione monoxime and 10 mM HEPES (pH 7.2). PLMneurons were visualized using an Axioplan 2 Nomarski Microscope (Zeiss)equipped with a SensiCam CCD camera (PCO Imaging Kelheim, Germany). CFP(427/10-25 excitation, 440 dichroic, 472/30-25 emission), YFP (504/12-25excitation, 520 dichroic, 542/27-25 emission), and FRET (427/10-25excitation, 440 dichroic, 542/27-25 emission) filters (Semrock), acolliminating emission port adapter (Photometrics), and the Slidebook5.0 software (Intelligent Imaging Innovations) were used to collect FRETdata. The CFP channel collects CFP emission after CFP excitation and theFRET channel collects YFP emission after CFP excitation. Analysis ofexported tiff files containing data from the FRET or CFP channel wasperformed using the ImageJ software (NIH). The FRET ratio was calculatedby (FRET_(PLM)−FRET_(bkgnd))/(CFP_(PLM)−CFP_(bkgnd)), where FRET_(PLM)and CFP_(PLM) are the mean fluorescent intensities in the FRET and CFPchannels of the PLM neuron and FRET_(bkgnd) and CFP_(bkgnd) are the meanfluorescent intensities in the FRET and CFP channels of a backgroundregion adjacent to the PLM neurons.

Statistical Analysis. Experimental data are presented as mean±standarderror of the mean (SEM). Significance of the differences between twodata sets was determined using the Student t test.

Results

To assess the activities of HBx in C. elegans, global expression of HBxwas induced under the control of the C. elegans heat-shock promoters(P_(hsp)HBx). Expression of HBx in C. elegans had deleterious effects,leading to a high percentage of embryonic lethality. Approximately 97%of P_(hsp)HBx transgenic embryos did not hatch and were invariablydeformed. Many contained large vacuoles that resembled necrotic cellsand raised discs characteristic of apoptotic cells. To determine thenature of HBx-induced cell death, the P_(hsp)HBx transgenes wasintroduced into animals defective in ced-3, which encodes a caspaseessential for apoptosis, or animals defective in mec-6, which isimportant for necrosis. A strong loss-of-function (lf) mutation inced-3(n2433) or mec-6(e1342) partially suppressed embryonic lethalitycaused by HBx overexpression, indicating that both apoptotic andnecrotic cell death contributes to lethality of HBx transgenic embryos.

To analyze the cell-killing activity of HBx, HBx was expressed in sixmechanosensory neurons (ALML/R, AVM, PVM, PLML/R) under the control ofthe mec-7 gene promoter (P_(mec-7)HBx). To aid in identification ofthese non-essential neurons, GFP was expressed under the control of themec-3 promoter (P_(mec-3)GFP), which drives gene expression in the samesix touch cells plus four other sensory neurons (PVDL/R and FLPL/R). Anintegrated transgene (smIs98) containing both P_(mec-7)HBx andP_(mec-3)GFP was used to assay killing of touch cells by HBx. Onaverage, 13-50% of touch cells in smIs98 animals underwent ectopic celldeath, with the two posterior PLM neurons showing most ectopic deaths(50%). PLM death was thus used in all subsequent genetic analysis. Somedying touch cells in smIs98 animals displayed an enlarged vacuolarmorphology characteristic of necrotic cells and some displayed theraised disc-like morphology of apoptotic cells. Consistently, cellkilling by HBx was partially suppressed by either ced-3(n2433) ormec-6(e1342) If mutations and eliminated by loss of both genes. Theseresults confirmed that HBx induces both apoptosis and necrosis in C.elegans.

Many genes involved in apoptosis and necrosis have been identified in C.elegans. In the apoptotic pathway, four key proteins, EGL-1 (similar tohuman BH3-only pro-apoptotic proteins), CED-9 (human Bcl-2 proteins),CED-4 (human Apaf-1), and CED-3 (human caspases), act sequentially tocontrol activation of apoptosis. In the necrotic pathway, severalconserved endoplasmic reticulum (ER) proteins involved in regulatingCa²⁺ homeostasis, including two Ca²⁺-binding proteins, CRT-1(calrecticulin) and CNX-1 (calnexin), and two Ca²⁺ channels, ITR-1(inositol trisphosphate receptor) and UNC-68 (uncoordinated, a ryanodinereceptor homolog), control release of Ca²⁺ from ER to cytosol inresponse to various necrotic insults. Ca²⁺ elevation in cytosol theninitiates necrosis through conserved Ca²⁺-dependent proteases, CLP-1(calpain family) and TRA-3 (transformer), and their downstream aspartylproteases (ASP-3 and ASP-4). Whether these key components of apoptoticand necrotic cell death pathways affect HBx-induced cell death wasexamined. Loss of egl-1, ced-3 or ced-4 partially suppressed HBx-inducedcell death (from 50% to 22-26% PLM death), indicating that HBx inducescell death partly through the apoptotic pathway. Likewise, loss ofcrt-1, cnx-1, itr-1, clp-1, tra-3, asp-3 or asp-4 partially suppressedHBx-induced cell killing (from 50% to 12-32% PLM death), indicating thatHBx induces cell death in part through the necrotic pathway.Importantly, loss of ced-3 and one of the components in the necroticpathway (mec-6, clp-1, itr-1, and crt-1) almost completely blockedectopic touch cell death induced by HBx, indicating that apoptosis andnecrosis account for virtually all cell death induced by HBx.

Like loss of egl-1, ced-4 or ced-3, a gain of function (gf) mutation(n1950) in ced-9 prevents most somatic apoptosis in C. elegans. However,unlike egl-1(lf), ced-4(lf) and ced-3(lf) mutations that partially blockHBx-induced cell death, ced-9(n1950gf) substantially completelyinhibited HBx-induced touch cell death and embryonic lethality.Moreover, a strong lf mutation in ced-9 (n2812) substantially completelysuppressed ectopic cell-killing induced by HBx in the ced-4(n1162)mutant background, which blocks massive ectopic cell deaths andembryonic lethality caused by ced-9(n2812) (25). These results indicatethat HBx induces both apoptotic and necrotic cell death through CED-9.

The ced-9(n1950gf) mutation results in substitution of Gly¹⁶⁹ by Glu inthe BH3-binding pocket of CED-9, which disrupts the binding of theBH3-only protein EGL-1 to CED-9 and prevents EGL-1-induced release ofthe pro-apoptotic CED-4 dimer from the CED-4/CED-9 complex tethered onthe surface of mitochondria. It is believed that HBx acts by binding toCED-9, thereby antagonizing its death inhibitory function. Whether HBxbinds CED-9 was examined in vitro using a glutathione S transferase(GST) fusion protein pull-down assay. GST-HBx specifically interactedwith CED-9 tagged with a six-histidine epitope but not with the mutantCED-9(G169E) protein. Interestingly, HBx contains a sequence (residues116-132) that is distantly related to the BH3 motif of manypro-apoptotic BH3-only proteins. The possibility that the interaction ofHBx with CED-9 occurs through this motif was tested by altering twoamino acids (Gly¹²⁴ to Leu and Ile¹²⁷ to Ala) in HBx that are conservedamong BH3-only proteins and that face CED-9 in a structural model of theHBx/CED-9 complex. G124L substitution markedly reduced and I127Asubstitution compromised the binding of HBx to CED-9 in vitro.Alteration of both residues (G124L; I127A) abolished association of HBxwith CED-9. Consistently, in an in vitro CED-4 releasing assay, HBx butnot the HBx(G124L; I127A) mutant, was able to release CED-4 from theGST-CED-9/CED-4 complexes tethered to Glutathione resin. When HBx(G124L)or HBx(I127A) was expressed under the control of the mec-7 promoter,both mutant proteins displayed reduced cell killing activity, while thedouble mutant, HBx(G124L; I127A), induced minimal ectopic touch celldeath. Similarly, HBx(G124L; I127A) did not cause embryonic lethality inC. elegans like HBx when expressed under the control of the heat-shockpromoters. These in vitro and in vivo results indicate that associationof HBx with CED-9 is required for HBx to induce ectopic cell killing inC. elegans.

To characterize further the interaction between HBx and CED-9,structural modeling of the HBx/CED-9 complex was performed using thepublished EGL-1/CED-9 complex structure, replacing the EGL-1 BH3 helixwith the putative BH3 motif of HBx. In the modeled HBx/CED-9 structure,Glu¹²⁵ and Glu¹²⁶ of HBx are in the vicinity of the Gly¹⁶⁹ residue ofCED-9. The replacement of Gly¹⁶⁹ by a bulky, negatively charged Glu inthe ced-9(n1950gf) mutant is expected to cause a steric clash and/orcharge repulsion with either Glu¹²⁵ or Glu¹²⁶ or both, disrupting theinteraction between HBx and CED-9. Two single Glu to Ala substitutions(E125A and E126A) in HBx was generated and tested whether residues witha neutral, smaller side chain may alleviate steric clash or chargerepulsion and restore binding of HBx to CED-9(G169E). Although neithermutation affected binding of HBx to wild-type CED-9, E125A, but notE126A, specifically restored binding of HBx to CED-9(G169E) in vitro. Invivo, both HBx(E125A) and HBx(E126A) caused ectopic touch cell death anda high percentage of embryonic lethality in wild-type animals like HBxwhen expressed under the control of the mec-7 and the heat-shockpromoters, respectively. However, only HBx(E125A), but not HBx orHBx(E126A), induced robust killing of touch cells and embryo inced-9(n1950) animals. These results correlate with the observation thatHBx(E125A), but not HBx or HBx(E126A), bound CED-9(G169E) in vitro.Together, they provide strong evidence that HBx induces cell death in C.elegans by directly interacting with CED-9.

The functions of CED-9 and Bcl-2 in cell death regulation are highlyconserved, and Bcl-2 can partially substitute for ced-9 to inhibitapoptosis in C. elegans. Therefore, whether HBx binds Bcl-2 familyproteins and if Bcl-2 can substitute for CED-9 in mediating HBx-inducedcell killing in C. elegans was tested. It was found that GST-HBxspecifically interacted with human anti-apoptotic proteins Bcl-2 andBcl-xL, but not with the pro-apoptotic protein Bax. The binding of HBxto Bcl-2 and Bcl-xL was markedly reduced by the G124L and I127Amutations, indicating that HBx also interacts with Bcl-2 and Bcl-xL invitro through its BH3-like motif.

HBx was unable to induce touch cell death in ced-9(n1950) animals owingto its inability to bind CED-9(G169E). This suppression of HBx-inducedcell death was completely reversed by expression of wild-type CED-9(P_(mec-7)CED-9) in smIs98; ced-9(n1950) animals. Importantly,expression of human Bcl-2 in touch cells (P_(mec-7)hBcl-2) alsocompletely restored HBx-induced cell killing in smIs98; ced-9(n1950)animals, indicating that HBx interactions with Bcl-2 and CED-9 and theensuing signaling mechanisms are conserved.

Since HBx acts through CED-9 to induce necrosis and because cytosolicCa²⁺ increase is essential for activation of necrosis in C. elegans,whether HBx targets CED-9 to affect intracellular Ca²⁻ was examined. AC. elegans strain carrying an integrated transgene (bzIs17) thatexpresses a cameleon Ca²⁺ sensor under the control of the mec-4 genepromoter was used to monitor intracellular Ca²⁺ in six touch cellsthrough the fluorescence-resonance energy transfer (FRET) assay. In thisassay, the FRET ratio (defined as the fluorescence intensity in the FRETchannel divided by the intensity in the CFP channel) is indicative ofrelative cytosolic Ca²⁺ concentrations. bzIs17 animals were treated withthapsigargin, which inhibits the ER ATPase that pumps Ca²⁺ from thecytosol into the ER and is expected to result in elevation of cytosolicCa²⁺. Indeed, the FRET ratio in PLM neurons of thapsigargin-treatedanimals was significantly higher than that of untreated orvector-treated animals. To assess the impact of HBx expression oncytosolic Ca²⁺ in living cells, the asp-3(tm4559) mutation was used toblock most of the touch cell deaths (from 50% missing PLMs to 12%)induced by an integrated P_(mec-7)HBx transgene (smIs451). Loss of asp-3on its own did not alter cytosolic Ca²⁺, as the FRET ratios of bzIs7 andbzIs17; asp-3(tm4559) animals were almost identical. However, in bzIs17;asp-3(tm4559); smIs451 animals, a 40% increase in the FRET ratio wasobserved, indicating that HBx expression in touch cells caused asignificant increase in cytosolic Ca²⁺. Importantly, the HBx-inducedCa²⁺ increase was obliterated by the ced-9(n1950) mutation, whichprevents HBx binding to CED-9. Therefore, it appears HBx directlytargets CED-9 to induce cytosolic Ca²⁺ increase in C. elegans.

CED-9 has been shown to localize to the outer membrane of mitochondriathrough its C-terminal transmembrane (TM) domain. A CED-9 mutant(CED-9ΔTM) lacking this TM domain fails to localize to mitochondria andis found in the cytoplasm. Interestingly, expression of CED-9ΔTM stillpartially rescues defects in apoptosis and embryonic lethality inced-9(lf) animals, indicating that mitochondrial localization is notessential for CED-9 to inhibit apoptosis in C. elegans. Whethermitochondrial localization is important for CED-9 to mediate HBx-inducedcell killing was examined. Expression of CED-9ΔTM in touch cells(P_(mec-7)CED-9ΔTM) only restored PLM killing in smIs98; ced-9(n1950)animals to 7-9%, compared to 46-52% PLM killing caused by expression ofwild-type CED-9. These results indicate that mitochondrial localizationis critical for CED-9 to mediate HBx-induced cell killing.

Because mitochondrial permeability transition (MPT) has been implicatedin mediating HBx-induced Ca²⁺ increase, whether MPT affects HBx-inducedcell killing and Ca²⁺ increase in C. elegans was tested. smIs98 animalswere treated with cyclosporin A (CsA), a peptide that desensitizes MPTby inhibiting the activity of the mitochondrial cyclophilin D, theessential regulatory component of MPT. In smIs98 animals that normallylost 50% of PLM neurons, CsA treatment reduced the percentage of missingPLM to 15%. Similarly, a deletion mutation (tm4171) in cyn-1, whichencodes the C. elegans cyclophilin D homolog, reduced the percentage ofmissing PLM in smIs98 animals to 17%. These results indicate that MPT islikely involved in HBx-induced cell killing. Moreover, loss of ced-3enhanced suppression of HBx-induced cell killing by CsA (from 15% PLMdeath in CsA-treated animals to 4% PLM death, whereas loss of eitherclp-1 or mec-6 did not. These results suggest that MPT is critical forHBx-induced necrotic cell death and provide the first report that MPT isimportant for necrosis in C. elegans.

Whether MPT is involved in HBx-induced cytosolic Ca²⁺ increase wasexamined. The FRET ratio of bzIs17; asp-3(tm4559); smIs451 animalstreated with CsA was reduced to the level seen in bzIs17; asp-3(tm4559)animals, indicating that HBx-induced cytosolic Ca²⁺ increase wascompletely suppressed by CsA. Therefore, HBx-induced Ca²⁺ increase islikely mediated by MPT, which is consistent with observations from humancells.

To identify targets or effectors of HBx-induced cell killing, a geneticscreen was conducted to isolate suppressors of the embryonic lethalityphenotype caused by global expression of HBx. To facilitateidentification of true HBx suppressors, the screen was performed insmIs98 animals that co-expressed HBx and GFP under the control of theheat-shock promoters (P_(hsp)HBx and P_(hsp)GFP). P_(hsp)GFP was used toeliminate mutations that affected transcription from the heat-shockpromoters, while smIs98 provided a secondary screen for true suppressorsof HBx-induced cell killing. From a screen of approximately 20,000haploid genomes, 31 HBx-induced death suppressors were isolated, whichwere named hids mutations. Most hids mutations not only suppressedembryonic lethality caused by global expression of HBx (P_(hsp)HBx) butalso reduced or blocked ectopic neuronal death in smIs98 animals,suggesting that they affect either the apoptotic or the necrotic pathwayor both. Indeed, one mutation (sm250) failed to complement clp-1(tm690)and is a nonsense allele of clp-1 (Trp²⁹⁵ to Opal). Another hids mutant(sm221) failed to complement mec-6(e1342) and is a missense allele ofmec-6 (Gly⁷⁷ to Ser). The third hids mutant (sm224) had no apoptoticcell corpse and is a nonsense allele of ced-4 (Glu³⁸³ to Ochre). Thesefindings indicate that the genetic suppressor screen successfullyidentified genes involved in HBx-induced apoptosis and necrosis and is apowerful tool to identify new apoptosis and necrosis genes andadditional effectors or targets of HBx.

Discussion

HBV infects more than 350 million people and is the leading cause ofliver disease and HCC worldwide. As the key pathogenic and oncogenicprotein encoded by HBV, HBx presumably interacts with host factors topromote virus replication and various pathogenic consequences, such asliver cell death and HCC. However, the host targets of HBx and itsmechanisms of action remain elusive, partly due to the lack of asuitable animal model amenable to genetic analysis, inconsistenciesoften associated with cell culture studies, and the genetic complexityof mammalian systems (e.g., mammals have at least six Bcl-2-like celldeath inhibitors). These technical hurdles have prevented definitiveidentification of HBx cellular targets in the last three decades.Therefore, development of a simpler animal model where HBx host factorsand downstream effectors can be identified and verified through powerfulmolecular genetic approaches becomes imperative for understanding HBxfunctions and treating HBV patients.

The present inventors have discovered that C. elegans, with its muchsimpler genome (only one Bcl-2 homolog) and powerful genetic tools,presents one such animal model. The present inventors' finding thatexpression of HBx in C. elegans induces both apoptosis and necrosis,which mimics one of the early cellular events following liver infectionby HBV, leads to systematic genetic dissection of HBx-induced cell deathpathways that involve highly conserved cell death regulators andexecutors. These include key regulators of apoptosis (CED-9, CED-4, andCED-3) and critical components in the necrosis pathway, especially thoseinvolved in regulating Ca²⁺ signaling, including ER Ca²⁺ bindingproteins and channels (CRT-1, CNX-1, UNC-68 and ITR-1), Ca²⁺-dependentproteases (CLP-1 and TRA-3), and MPT, many of which previously have notbeen implicated in HBV or HBx-induced pathogenesis. MPT, however, hasbeen implicated in HBx-induced cell killing and HBV replication inhumans. CED-9, a key apoptosis regulator, was discovered unexpectedly tobe a host target of HBx in cell death and Ca²⁺ signaling in C. elegans,which led to identification of human Bcl-2 proteins as conserved hosttargets of HBx-mediated Ca²⁺ stimulation and HBV replication in humanhepatocytes. These findings demonstrate the validity of the C. elegansmodel for studying HBV and HBx. The HBx suppressor screen has identifiedimportant regulators of both cell death pathways and still has thepotential to identify new targets or effectors of HBx as well as newapoptosis and necrosis genes.

Importantly, the cell killing activity of HBx is dependent on CED-9, aBcl-2 homolog and a key cell death inhibitor, because a gain-of-functionmutation (G169E) in the BH3-binding pocket of CED-9 blocks HBx-inducedcell death in C. elegans. In vitro, HBx interacts with CED-9, but notwith CED-9(G169E), through its BH3-like motif. Alteration of twoconserved residues in the BH3-like motif of HBx abolishes binding of HBxto CED-9 and HBx's cell killing activity. A compensatory mutation(E125A) in the BH3-like motif of HBx that restores binding of HBx toCED-9(G169E) allows HBx to kill efficiently in ced-9(n1950gf) animals.These in vitro and in vivo results establish that HBx interacts directlywith CED-9 through its BH3-like motif to induce cell killing, and thatCED-9 is the bona fide cellular target of HBx. Importantly, HBxinteracts with Bcl-2 and Bcl-xL, two human anti-apoptotic CED-9homologs, through the same BH3-like motif in human hepatocytes and thisinteraction is critical for HBx-induced cytosolic Ca²⁺ elevation, celldeath, and HBV viral replication. These results and the finding thatBcl-2 can fully substitute for CED-9 in C. elegans to mediateHBx-induced cell killing suggest that HBx acts through conserved hosttargets (Bcl-2 family members) and conserved signaling pathways toinduce cytosolic Ca²⁺ elevation, cell killing, and other cellular andviral events. They also demonstrate the unique advantage of the C.elegans genetic system for unambiguous determination of in vivo proteininteraction and target identification, a daunting task in complexmammalian systems.

One of the signaling events associated with HBx expression inhepatocytes is elevation of cytosolic Ca²⁺, which is critical for HBVreplication, transcription and core assembly and is involved inactivation of cell death and several other signaling pathways. Althoughthe cellular target of HBx-mediated calcium stimulation is unknown, HBxhas been proposed to target mitochondria to effect permeabilitytransition, which plays an important role in regulating intracellularCa²⁺ homeostasis. In this study, the present inventors have shown thatHBx interacts directly with CED-9, a mitochondrial protein, to increasecytosolic Ca²⁺, which then triggers activation of necrosis in C. elegansthrough Ca²⁺-dependent proteases (CLP-1 and TRA-3). CED-9-dependent Ca²⁺elevation and necrosis induced by HBx can both be suppressed by CsA, aninhibitor of MPT. These results are consistent with the observations inhuman cells that HBx acts through MPT to control intracellular calcium,cell death, and HBV replication and indicate that CED-9 is the cellulartarget of HBx in elevating intracellular Ca²⁻. Since Bcl-2 associateswith HBx in HBV-infected hepatocytes, can substitute for CED-9 inmediating HBx-induced cell killing in C. elegans, and has beenimplicated in regulating mitochondria permeability transition, Bcl-2 andBcl-xL, both of which are mitochondrial proteins, are likely targeted byHBx during HBV infection to alter Ca²⁺ signaling. The induced cytosolicCa²⁺ increase then triggers activation of multiple viral and hostevents, including HBV replication, assembly and cell death. Therefore,targeting the BH3-like motif of HBx to prevent HBx binding to Bcl-2family proteins could be a new and ideal therapeutic strategy fortreating HBV-related liver disorders without perturbing host cellsignaling pathways.

Example 3

Microscale liquid culture of C. elegans. Peptaibol TK was mixed with theS-medium to form 70% peptaibol TK concentration solution. This mixturewas placed in 96-well culture plate, along with worms and pure S-medium(wormbook, maintenance of C. elegans) as control. Centrifugalsedimentation of HB101 was suspended by culture liquid as C. elegansfood supply in 1:1 volume rate.

Embryo/germline cell corpse counting assay. Embryo and germline cellcorpse were counted under DIC field of Zeiss Nomarski microscope. Forembryo cell corpse counting, late L4 worms were transferred intomicroscale liquid culture and their egg lay in 24 hours were scored forcell corpses. For germline cell corpse counting, middle L4 worms weretransferred into microscale liquid culture and counted their germlinecell corpse at 24 hours and 48 hours post liquid culture. The allelesused in cell corpse counting assay were egl-1(n3082), ced-9(n1950),ced-4(n1162) and ced-1(e1735).

Gonad dissection. Gonad dissection was performed on smIs203 worms, whichcarry an integrated CED-4::GFP transgene. Worms were dissected on commonglass slide. After anaesthetizing worms with 10 μL of 30 μM sodiumazide, more 30 μL M9 was added for dilution. To dissect the gonad, theworm's head was placed between two syringe needles and decapitate bymoving needles in a scissors motion. Then, released gonad was observedafter covering with cover glass.

Results

In order to understand how peptaibol TK selectively promotes apoptosisin human cancer cells, apoptosis in C. elegans was tested using amixture of peptaibol TK VI (SEQ ID:NO 1), peptaibol TK VII (SEQ ID:NO 2)and peptaibol TK VIII (SEQ ID:NO 3). See FIG. 1 a. C. elegans animalswere cultured in 96-well plates in S medium containing 20 μM peptaibolTK and the number of apoptotic cell corpses in C. elegans embryos andgermline were counted. To sensitize the cell death assay, ced-1(e1735)animals, in which engulfment of apoptotic cells is blocked, were used toallow easy scoring of apoptotic cells. It was found that compared to thebuffer control peptaibol TK treatment caused significantly moreapoptotic cell corpses in both ced-1(e1735) embryos and germline (FIGS.1a-c ), indicating that peptaibol TK can induce ectopic apoptosis in C.elegans.

To investigate how peptaibol TK induces ectopic cell death, the geneticrequirement for peptaibol TK-induced cell death was examined. Thecentral cell killing pathway in C. elegans is mediated by a negativeregulatory cascade, in which the cell death initiator EGL-1 induces celldeath by antagonizing the activity of CED-9, a cell death inhibitor,which allows the caspase activator CED-4 to activate the cell killingcaspase CED-3 (FIG. 1f ). It was found that a strong loss-of-function(lf) mutation in ced-4(n1162) or ced-3(n2433) completely blockedpeptaibol TK-induced ectopic cell death in both ced-1(e1735) embryos andgermline (FIG. 1d ). These results indicate that peptaibol TK actsupstream of or in parallel to ced-4 to induce apoptosis. Similarly, astrong if mutation in egl-1(n3082) completely blocked peptaibolTK-induced ectopic cell death in ced-1(e1735) embryos (FIG. 1d ).Interestingly, a gain-of-function (gf) mutation in ced-9 (n1950) causesa Gly169 to Glu substitution in the BH3-motif binding pocket of theCED-9 protein that blocks binding of the BH3-only protein EGL-1 to CED-9and thus EGL-1 induced release of CED-4 from the inhibitory CED-4/CED-9complex tethered on the outer membrane of mitochondria. Likeegl-1(n3082), ced-9(n1950) blocked peptaibol TK-induced ectopic celldeath in ced-1(e1735) embryos (FIG. 1d ). These results indicate thatpeptaibol TK acts upstream of or in parallel to CED-9 and could targetCED-9 directly to induce apoptosis.

Since release of CED-4 from the inhibitory CED-4/CED-9 complex is a keyevent in C. elegans apoptosis, whether peptaibol TK promotes CED-4release to induce apoptosis was investigated. It has been reported thatduring apoptosis CED-4 disassociates from mitochondria and translocatesto the perinuclear region in apoptotic cells. A low-copy integratedtransgene (smIs203) carrying a translational CED-4::GFP fusion under thecontrol of the endogenous ced-4 promoter was generated. In dissectedgonad from smIs203 animals, CED-4::GFP displayed predominantly punctatecytoplasmic staining characteristic of mitochondria localization.Staining with MitoTracker Red, a mitochondria-specific dye, confirmedthat CED-4::GFP co-localized with MitoTracker Red to mitochondria.Treatment of peptaibol TK resulted in loss of cytoplasmic staining andappearance of GFP rings, which indicate translocation of CED-4::GFP frommitochondria to the nuclear membrane. These results indicate thatpeptaibol TK can promote apoptotic CED-4 release in vivo.

Whether peptaibol TK can promote CED-4 release in vitro was alsoexamined. In this experiment, peptaibol TK was incubated with theCED-4/CED-9 complex in the presence or absence of limited amount ofEGL-1, which alone was barely able to release CED-4 from the CED-4/CED-9complex. Although peptaibol TK alone was incapable of causing release ofCED-4, it greatly enhanced the release of CED-4 in combination withEGL-1. Thus, peptaibol TK can cooperate with EGL-1 to promote release ofCED-4 from the CED-4/CED-9 complex and thus apoptosis.

The enhanced EGL-1-induced release of CED-4 from the CED-4/CED-9 complexby peptaibol TK was examined using the gel-filtration assay. When 7 nmolof the CED-4/CED-9 complexes were loaded on the Superdex 200 column, thecomplex was eluted in a single peak. When the same amount of CED-4/CED-9complexes were pre-incubated with 12 nmol of EGL-1 and then loaded onthe Superdex column, a minor second peak consistent with the size ofCED-4 tetramer was observed, indicating limited amount of CED-4 releasefrom the CED-4/CED-9 complex. Although pre-incubation of the same amountof CED-4/CED-9 complexes with 150 nmol of peptaibol TK did not cause anydetectable CED-4 release, pre-incubation of 150 nmol of peptaibol TK and150 nmol of EGL-1 with the CED-4/CED-9 complexes caused a major shift ofthe CED-4/CED-9 protein peak to the CED-4 tetramer peak, indicating agreatly increased release of CED-4 from the CED-4/CED-9 complexes. Takentogether, these in vitro CED-4 releasing results suggest that peptaibolTK promotes apoptosis by enhancing EGL-1-induced release of CED-4 fromthe inhibitory CED-4/CED-9 complex and are consistent with theobservations that peptaibol TK failed to induce cell death inegl-1(n3082) embryos (FIG. 1d ).

To probe how peptaibol TK enhances EGL-1-induced release of CED-4 fromthe CED-4/CED-9 complex, whether peptaibol TK interacts with EGL-1,CED-9, or both was tested using isothermal titration calorimetry (ITC)assays. When peptaibol TK was used to titrate CED-9, the well-correlateddifferential heating power and the molar ratio between peptaibol TK andCED-9 indicate a weak but detectable interaction with a binding affinity(K_(B)) at 4.26×10⁴ M⁻¹. By contrast, titration of EGL-1 with peptaibolTK showed scattered data points with small variations, which isindicative of background thermal fluctuations and no significantinteraction between EGL-1 and peptaibol TK. As expected, CED-9interacted with EGL-1 strongly, yielding a binding affinity of 4.04×10⁶M⁻¹. When CED-9 was pre-incubated with peptaibol TK before beingtitrated with EGL-1, a much stronger binding affinity (1.52×10⁸ M⁻¹) wasdetected, which is approximately 38 folds of that between CED-9 andEGL-1. Given that peptaibol TK didn't bind EGL-1, this great enhancementin binding affinity between EGL-1 and CED-9 is likely caused byinteraction of peptaibol TK with CED-9, which may induce an allostericchange in CED-9 that greatly enhances its binding to EGL-1. This is alsoconsistent with the observation that peptaibol TK enhances EGL-1-inducedrelease of CED-4 from the CED-4/CED-9 complex both in vitro and in vivo.

The Hepatitis B Virus X protein, HBx, has been shown to play a criticalrole in pathogenesis and neoplastic transformation of hepatocytes inHBV-infected patients. Interestingly, HBx interacts with highlyconserved host targets, the Bcl-2 family cell death inhibitors, througha BH3-like motif to promote intracellular calcium increase, leading toapoptosis, necrosis and viral replication. Since HBx induces bothapoptotic and necrotic cell death in C. elegans through interacting withCED-9, whether peptaibol TK similarly enhances HBx-induced cell death inC. elegans was tested using smIs98 animals, which carry an integratedtransgene that expresses HBx and GFP under the control of thetouch-cell-specific mec-7 and mec-3 gene promoters (P_(mec-7)HBx andP_(mec-3)GFP), respectively. In smIs98 animals treated with peptaibolTK, the percentage PLM touch cell death is 71%, which is significantlyhigher than (52%) that observed in smIs98 animals treated with buffercontrol (FIG. 2). On the other hand, peptaibol TK or buffer controltreatment of smIs3 animals, which carry an integrated transgenecontaining only P_(mec-3)GFP and without P_(mec-7)HBx, resulted in asimilar low percentage of PLM cell killing (FIG. 2). Similar resultswere obtained when P_(mec-7)HBx was present as an extrachromosomal arrayin bzIs8 animals, which carry an integrated transgene containingP_(mec-4)GFP that also label touch cells specifically with GFP (FIG. 2).These results indicate that peptaibol TK can enhance HBx-induced celldeath in C. elegans, just as it does in enhancing EGL-1-induced celldeath.

Because HBx promotes cell killing in C. elegans and in human hepatocytesthrough conserved host targets, the Bcl-2 family cell death inhibitors,whether peptaibol TK can promote increased cell killing inHBV-transfected human hepatocytes was tested. HepG2-N10 cells, which arehuman hepatic cells carrying an integrated copy of 140% head-to-tail HBVgenome (pHBV), and control HepG2 cells were treated with variousconcentration of peptaibol TK. Increasing concentrations (4, 8, 12, 16μM) of peptaibol TK did not seem to significantly alter the cell densityof the control HepG2 cells, but significantly reduced the cell densityof the HepG2-N10 cells, suggesting that peptaibol TK promotes increasedcell killing in HBV-transfected hepatic cells, but not in uninfectedHepG2 cells. The effect of peptaibol TK on C57BL/6 mice carrying anintegrated copy of pHBV and control C57BL/6 mice were tested throughintraperitoneal injection. Compared to the control C57BL/6 mice, HBVtransgenic mice are more sensitive to the peptaibol TK injection, as theserum Alanine Aminotransferase (ALT) levels, an indicator of liverdamage, are higher in HBV transgenic mice than the control mice sixhours post IP injection. These results provide preliminary supportingevidence that peptaibol TK promotes increased cell killing selectivelyin HBV-transfected hepatocytes.

Discussion

Similar to the situation in cultured cell, peptaibol TK induced celldeath in C. elegans through the apoptosis pathway. Genetic analysisindicated that peptaibol TK works cooperatively with EGL-1 to kill cellby apoptosis. To prove this hypothesis, experiment was performed whichshowed that peptaibol TK indeed promoted more CED-4 release in vivo andin vitro, where the EGL-1 is present, to enhance the apoptosis-inducingeffect. Further, ITC was used to investigate the minutiae of interactionprocess among peptaibol TK, EGL-1 and CED-9/CED-4 complex. Based on theexperimental data, it appears firstly, peptaibol TK binds to CED-9 ofCED-9/CED-4 complex in a thermodynamically unsteady manner. Peptaibol TKbinding is believed to change the conformation of CED-9, turning it intoa state that has a higher affinity for EGL-1 and thus greatlyfacilitating EGL-1-induced release of CED-4.

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter. All references cited herein are incorporated by reference intheir entirety.

What is claimed is:
 1. A method for treating hepatitis B infection in asubject, said method comprising administering to a subject in need ofsuch a treatment a composition (i) that is capable of inhibiting bindingof hepatitis B virus X protein to a Bcl-2 family member protein in saidsubject; (ii) that is capable of reducing the expression of Bcl-2familymember protein in said subject; or (iii) a combination thereof.
 2. Themethod of claim 1, wherein said Blc-2 family member protein comprisesBcl-2, Bcl-xL, or a combination thereof.
 3. The method of claim 1,wherein said composition comprises a binding inhibitor that is capableof inhibiting binding of hepatitis B virus X protein to said Bcl-2family member protein.
 4. The method of claim 3, wherein said bindinginhibitor is capable of interacting with Bcl-2 homology 3 (BH3)-likemotif of hepatitis B virus X protein or a Bcl-2 family member.
 5. Themethod of claim 1, wherein said composition comprises an expressioninhibitor that is capable of reducing the expression of Bcl-2 familymember protein.
 6. The method of claim 5, wherein said expressioninhibitor is a siRNA or a small RNA that is capable of reducing theexpression of Bcl-2, Bcl-xL, or a combination thereof.
 7. A method foridentifying a composition that is capable of treating hepatitis B virus(HBV) infection in a mammal, said method comprising determining theeffect of said composition in an interaction between HBV X protein andCED-9 protein of C. elegans, wherein modulation of the interactionbetween HBV X protein and CED-9 protein in the presence of saidcomposition is an indication that said composition is capable oftreating HBV infection in a mammal.
 8. A method for treating hepatitis Binfection in a subject, said method comprising administering to asubject in need of such a treatment a therapeutically effective amountof a composition comprising peptaibol TK.
 9. The method of claim 8,wherein said peptaibol TK comprises peptaibol TK VI (SEQ ID NO: 1),peptaibol TK VII (SEQ ID NO: 2), peptaibol TK VIII (SEQ ID NO: 3) or amixture thereof.